Plants synthesize a variety of hydrocarbons built up of isoprene units (C5H8), termed polyisoprenoids (Tanaka, Y. In Rubber and Related Polyprenols. Methods in Plant Biochemistry; Dey, P. M. and Harborne, J. B., Eds., Academic Press: San Diego, 1991; Vol. 7, pp 519–536). Those with from 45 to 115 carbon atoms, and varying numbers of cis- and trans- (Z- and E-) double bonds, are termed polyprenols, while those of longer chain length are termed rubbers (Charlwood et al., In Minor Classes of Terpenoids. Methods in Plant Biochemistry; Dey, P. M. and Harborne, J. B., Eds., Academic Press: San Diego, 1991; Vol. 7, pp 537–542). The synthesis of these compounds is carried out by a family of enzymes termed prenyltransferases, which catalyze the sequential addition of C5 isopentenyl diphosphate units to an initiator molecule (FIG. 1). In Hevea brasiliensis rubber, the C5 units are added in the cis-configuration, and thus the prenyltransferas(s) involved are termed cis- or Z-prenyltransferases.
Two distinct pathways for the synthesis of isopentenyl diphosphate (IPP) are now known to be present in living organisms (Lichtenthaler et al., Physiol. Plantarum 101:643–652 (1997)). In one pathway, which is confined in plants to plastids, glyceraldehyde 3-phosphate and pyruvate are precursors of IPP (Lichtenthaler et al., FEBS Letts. 400:271–274 (1997)). In the second (cytoplasmic) pathway, acetate is converted to IPP via the intermediate mevalonic acid (Newman, J. D., Chappell, J. Isoprenoid biosynthesis in plants: carbon partitioning within the cytoplasmic pathway. Crit Rev Biochem Mol Biol. 1999;34(2):95–106; Bach T J, Boronat A, Campos N, Ferrer A, Vollack K U, Mevalonate biosynthesis in plants. Crit Rev Biochem Mol Biol. 1999;34(2): 107–22). The latter pathway, the acetate/mevalonate pathway, has long been assumed to be the sole pathway operating in the rubber-synthesizing latex of Hevea brasiliensis. In this pathway, acetate is converted to IPP by the sequential action of the following six enzymes: acetyl-coA acetyltransferase, HMG-coA synthase, HMG-coA reductase, mevalonate kinase, phosphomevalonate kinase and mevalonate diphosphate decarboxylase (FIG. 2).
Of the minimum of six genes encoding the enzymes of this pathway in Hevea brasiliensis, only those for HMG-coA reductase have been cloned. Two cDNAs, encoding enzymes termed HMGR1 and HMGR2, were isolated using a heterologous hybridization probe, and genomic southern blotting confirmed the presence of at least two genes for HMG-coA reductase in the Hevea brasiliensis genome (Chye et al., Plant Mol. Biol. 16:567–577 (1991)). An EST homologous with HMGR1 was also identified in a Hevea brasiliensis latex library (Han et al., Tree Physiol. 20:503–510 (2000)). A gene encoding a third isoform of HMG-coA reductase in Hevea, termed HMGR3, has also been reported (Chye et al (1992) Plant Mol. Biol. 19: 473–484). Of the other five genes, although several have been identified in other plant species, no Hevea brasiliensis homologs have been identified or their genes isolated.
The initiator molecules used for the elaboration of polyprenols and rubbers are also derived from IPP, and are allylic terpenoid diphosphates such as dimethylallyldiphosphate (DMAPP), but more usually the C10 compound geranyl diphosphate (GPP), the C15 compound farnesyl diphosphate (FPP) or the C20 compound geranylgeranyl diphosphate (GGPP) (FIG. 1). DMAPP is generated from IPP by the action of an isomerase enzyme termed IPP isomerase. Genes encoding this enzyme have been isolated from a number of species, including Hevea brasiliensis (Oh et al., J. Plant Physiol. 157:549–557 (2000)). The allylic diphosphates GPP, FPP and GGPP are synthesized by trans- or E-prenyltransferases, using DMAPP and IPP. Genes encoding the enzymes which synthesize these allylic terpenoid diphosphates have been cloned from a number of organisms, including plants (McGarvey et al., Plant Cell 7:1015–1026 (1995); Chappell, J., Annu. Rev. Plant Physiol. Plant Mol. Biol. 46:521-547 (1995)). All of these gene products condense isoprene units in the trans-configuration.
There are several suggested functions for plant polyisoprenoids. Terpenoid quinones are most likely involved in photophosphorylation and respiratory chain phosphorylation. Rubbers have been implicated in plant defense against herbivory, possibly serving to repel and entrap insects and seal wounds in a manner analogous to plant resins. The roles of the C45–C115 polyprenols remain unidentified, although as with most secondary metabolites they too most likely function in plant defense. Short-chain polyprenols may also be involved in protein glycosylation in plants, by analogy with the role of dolichols in animal metabolism.
The problem to be solved is to provide a pathway for the synthesis of poly-cis-isoprenoids (rubbers). Applicants have solved the stated problem by the discovery of unknown genes (except for HMG-coA reductase) for each step of the acetate/mevalonate biosynthetic pathway in latex of Hevea brasiliensis. More specifically, the instant invention pertains to the identification and characterization of EST sequences from Hevea brasiliensis latex encoding acetyl-coA acetyltransferase, HMG-coA synthase, mevalonate kinase, phosphomevalonate kinase and mevalonate diphosphate decarboxylase. A shorter variant of putative acetyl co-A acetyltransferase has also been identified.